Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited disease of the kidney, caused by alterations of polycystin 1 and 2 (PC1 and PC2). ADPKD is characterized by multiple fluid-filled cysts that affect the structure and function of the kidney, leading to chronic renal failure. Because of its essential function in the vectorial movement of salt and water in the kidney, the Na,K-ATPase has been the focus of investigation to understand the pathophysiology of ADPKD. However, the precise role of the Na,K-ATPase in ADPKD remains unknown. Interestingly, besides its activity as an ion transporter, the Na,K-ATPase also functions as the receptor for ouabain. Ouabain is a well characterized hormone known to trigger signaling events that induce changes in metabolism, motility and growth in several cell types. Surprisingly, we have found that the Na,K-ATPase of epithelial cells from kidney cysts of patients with ADPKD (ADPKD cells) exhibit an abnormal increase in the affinity for ouabain. Moreover, we have found that ouabain, in amounts similar to those circulating in blood, stimulates cell growth in ADPKD cells. This effect is not found in normal human kidney epithelial cells (NHK cells), which have a lower affinity for the hormone. Furthermore, ouabain enhances ADPKD cell apoptosis, in a manner that does not parallel the increase in cell proliferation; decreases the adhesion properties of the cells; and enhances their ability to form cysts in culture. All these events are hallmarks of ADPKD cystogenesis. Accordingly, we have found that ouabain exacerbates renal cyst development in a mouse model of ADPKD, both in vitro and in vivo. Altogether, these results suggest that ouabain is a novel factor that promotes renal cyst formation and progression. At present, the role and mechanisms by which ouabain mediates cyst formation in ADPKD cells are unknown. Our overall goal in this research is to understand how Na,K-ATPase-ouabain signaling affects ADPKD. Our central hypothesis is that, due to their high ouabain sensitive phenotype, ADPKD cells are more susceptible to circulating levels of the hormone; which abnormally stimulates Na,K-ATPase signaling, to cause hyperplasic growth, fluid secretion and changes in the adhesion properties and motility of the cells. Experiments are designed to determine the mechanisms of ouabain stimulated Na,K-ATPase signaling in cyst formation of ADPKD cells, the intracellular pathways responsible for ouabain action on the cells, and the determinants of the abnormal ouabain affinity of ADPKD cells. The results will have an important positive impact, because they will help define the molecular mechanisms involved in cyst development and progression in ADPKD, and will contribute in the future to develop approaches that will target Na,K-ATPase signaling to control the disease. PUBLIC HEALTH RELEVANCE: The Na,K-ATPase is an essential molecule with important ion transport and signaling functions in the cell. The present application proposes to investigate the role and mechanism(s) of action of the Na,K-ATPase as a receptor and signal transducer of the effects of the hormone ouabain in the proliferation, fluid secretion and cyst formation of renal epithelial tubular cells from kidneys of patients with autosomal dominant polycystic kidney disease (ADPKD). This is important to define the molecular mechanisms involved in cyst development and progression in ADPKD, and will contribute in the future to develop approaches that will target Na,K-ATPase signaling to control the disease.